The principle behind vaccination is to induce an immune response in the host thus providing protection against subsequent challenge with a pathogen. This may be achieved by inoculation with a live attenuated strain of the pathogen, i.e. a strain having reduced virulence such that it does not cause the disease caused by the virulent pathogen.
Clasically, live attenuated vaccine strains of bacteria and viruses have been selected using one of two different methodologies. Mutants have been created either by treatment of the organism using mutagenic chemical compounds or by repeated passage of the organism in vitro. However, use of either method gives rise to attenuated strains in which the mode of attenuation is unclear. These strains are particularly difficult to characterise in terms of possible reversion to the wild type strain as attenuation may reflect single (easily reversible) or multiple mutation events. Furthermore, it is difficult to obtain such strains having optimum immunogenic properties because of multiple mutation events, and multiple strains may need to be used to provide protection against the pathogen.
Using modern genetic techniques, it is now possible to construct genetially defined attenuated bacterial strains in which stable attenuating deletions can be created. A number of site directed mutants of Salmonella have been created using this type of technology (2, 4, 5, 9, 12, 16, 17, 18). Mutations in a large number of genes have been reported to be attenuating, including the aro genes (e.g. aroA, aroC, aroD and aroE), pur, htrA, ompR, ompF, ompC, galE, cya, crp and phoP.
Salmonella aroA mutants have now been well characterised and have been shown to be excellent live vaccines against salmonellosis in several animal species. In addition, in order to reduce the chances of a reversion to virulence by a recombination event, mutations have been introduced into two independent genes such as aroA/purA and aroA/aroC. Identical mutations in host adapted strains of Salmonella such as S. typhi (man) and S. dublin (cattle) has also resulted in the creation of a number of candidate single dose vaccines which have proved successful in clinial (8, 11) and field trials (10).
A Salmonella typhimurium strain harboring stable mutations in both ompC and ompF is described in Chatfield et al (1991, ref. 21). When administered orally to BALB/c mice the strain was attenuated, with the 50% lethal dose (LD50) reduced by approximately 1,000-fold. However, the intravenous LD50 was reduced only by approximately 10-fold, demonstrating the importance of the porins in confering on the bacteria the ability to infect by the oral route.
Expression of the ompC and ompF genes is regulated by ompR. Pickard et al (1994, ref. 13) describes the cloning of the ompB operon, comprising the ompR and envZ genes, from a Salmonella typhi Ty2 cosmid bank and characterisation by DNA sequence analysis. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The S. typhi ompR mutants displayed a marked decrease in ompC and ompF porin expression as demonstrated by examination of outer membrane preparations. It was also shown that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi. 
In animal studies, attenuated S. typhimurium has been used as a vehicle for the delivery of heterologous antigens to the immune system (3, 6, 15). This raises the potential of the development of multivalent vaccines for use in man (7).